An immunoassay has been widely used when measuring a substance to be examined (analyte) in a sample, particularly an analyte present trace amounts in a biological sample. Most of such trace analytes are generally contained in the sample only in an amount of a .mu.g/ml unit or less. For example, there is a detectability limitation in an immunodiffusion or laser nephelometer method, wherein a complex produced from an antigen-antibody reaction is directly measured. Therefore, such trace analytes in the biological sample may be measured more accurately by a method, wherein one of the antigen or antibody is labeled with a suitable substance and a signal originating therefrom is detected, namely, a label-immunoassay, or the like. The label-immunoassay may be carried out in many ways. For example, there may be mentioned a forward sandwich assay or a delayed one-step sandwich assay. The one-step sandwich assay can be carried out using the following steps: (1) a sample is brought into contact with an insoluble carrier covered with a first immunological partner of an analyte; (2) after a washing treatment is optionally carried out, the resulting first immunological partner-analyte-complex carried on the insoluble carrier is brought into contact with a labeled second immunological partner; (3) the resulting complex of the insoluble carrier-first immunological partner-analyte-labeled second immunological partner, and a portion not containing said complex are separated; and (4) a signal originating from the label contained in one of the complex or the portion without said complex is detected. A one-step sandwich assay may also be carried out using the following steps: (1) a sample is brought into contact with an insoluble carrier covered with a first immunological partner of an analyte, and at the same time with a labeled second immunological partner; (2) the resulting complex of the insoluble carrier-first immunological partner-analyte-labeled second immunological partner, and a portion not containing said complex are separated; and (3) a signal originating from the label contained in one of the complex or the portion without said complex is detected. A reverse sandwich assay may also be carried out using the following steps: (1) a sample is brought into contact with a labeled first immunological partner of an analyte; (2) the resulting labeled first immunological partner-analyte-complex is brought into contact with a second immunological partner carried on an insoluble carrier; (3) the resulting complex of the labeled first immunological partner-analyte-second immunological partner-insoluble carrier, and a portion not containing said complex are separated; and (4) a signal originating from the label contained in one of the complex or the portion without said complex is detected. An immunoinhibition method may also be carried out using the following steps: (1) a sample is brought into contact with a labeled first immunological partner (preferably a labeled monoclonal antibody in case of an antibody) of an analyte, and then with a substance which shows a function same as that of the analyte and is carried on an insoluble carrier, or an immunological partner to the analyte, said partner being carried on an insoluble carrier; (2) the resulting immunological partner-substance showing the function same as that of the analyte-insoluble carrier complex, and a portion not containing said complex are separated; (3) and a signal originating from the label contained in one of the complex or the portion without said complex is detected. A competitive method may also be carried out using the following steps: (1) a sample is competitively brought into contact with an insoluble carrier covered with an immunological partner to an analyte, and a substance which shows a function the same as that of the analyte; (2) the resulting insoluble carrier-immunological partner-substance showing the function the same as that of the analyte complex, and a portion not containing said complex are separated; (3) and a signal originating from the label contained in one of the complex or the portion without said complex is detected.
Further, the label-immunoassay may be classified, in view of the label used, under, for example, an EIA wherein an enzyme is used as a label, an immunoagglutination method wherein erythrocytes or latex particles are used as a carrier, and the resulting aggregates are visually observed, RIA wherein an isotope is used as a label and the like. In these methods, the amount of the complex produced is increased with the contacting time of the sample with the immunological partner to the analyte. Therefore, the contacting of the sample with the immunological partner is lengthily continued to reach the equilibrium state, namely until the amount of the complex produced is not changed.
The RIA method requires particular equipment and has a problem of radioactive wastes, and thus, has gradually been replaced with the EIA method or the like. Further, when a blood sample is used in all the above-mentioned conventional methods, the collected whole blood sample was not used as they are, but a serum or plasma sample was prepared from the collected whole blood, and then an assay was carried out. When a whole blood sample containing insoluble components such as hemocytes is used in general methods other than the above methods, the insoluble components would possibly interfere with the value measured. For example, when the lights as detecting signals originating from the labels are measured in a visible light range in a homogeneous assay wherein B/F separation is not carried out, a value with a positive error may be obtained. In agglutination, an accurate determination cannot be carried out, because substances, such as hemocytes, which produce turbidity independently of the desired agglutination exist. On the other hand, the B/F separation assay may be conducted by reacting a whole blood sample with an antibody immobilized on an insoluble carrier, washing out the whole blood, and then reacting with a labeled antibody or the like. However, the result of the above assay is different from that of an assay using a serum sample, because there exist a lot of hemocyte components in the whole blood. Further, the results cannot be adjusted by multiplying a constant coefficient, because each individual has a different amount of hemocytes. Therefore, the result obtained from a whole blood sample should be adjusted by measuring a hematocrit value of the blood examined so as to obtain a diagnostic criterion from the whole blood sample. Taking into account such troublesome procedure, a serum or plasma sample prepared from a whole blood sample was used even in an assay wherein B/F separation was carried out.
Many attempts to avoid the influence of the insoluble components have been made. For example, Japanese Examined Patent Publication (Kokoku) No. 02-51150 discloses a method to avoid the influence of the insoluble components by adding a hemolyzing agent to whole blood before agglutination. Japanese Unexamined Patent Publication (Kokai) No. 01-237454 discloses a method to enhance detectability by enzymatic digestion or exposure to a mild acid to remove interfering substances and expose binding sites in a whole blood sample. Japanese Unexamined Patent Publication (Kokai) No. 01-165964 discloses a method to reduce the influence of the insoluble components by treating a whole blood with neuraminidase to liberate latent tumor-associated antigens. Further, as an attempt to conduct measurement without removing insoluble components, a method for detecting hepatitis virus by a dried filter blood is disclosed in Japanese Unexamined Patent Publication (Kokai) No. 64-63868. In this method, a filter paper stained with blood is air-dried, the dried filter paper is cut up to a piece with a predetermined surface area, and the piece, a buffer to extract the blood components contained the piece, and beads coated with antibodies are reacted for about 20 hours. However, the above method requires the pretreatment procedures of the air-drying of the blood on the filter paper and cutting of the dried paper. The above method does not use a whole blood sample as it is, but uses the blood components extracted from the filter paper.
In addition to the above-mentioned methods using immunoreaction in liquid, an analyte may be detected using a whole blood sample, by a method using a dry immunoassay element called dry chemistry [Japanese Unexamined Patent Publication (Kokai) No. 01-112159], a method using so-called biosensor [Japanese Unexamined Patent Publication (Kokai) No. 04-502671], or a method using immunochromatography [Japanese Unexamined Patent Publication (Kokai) No. 06-94718].
In the above-mentioned conventional methods wherein immunoreaction is carried out in liquid, an additional procedure (pretreatment) is required before the immunoreaction, and thus the assay procedure becomes troublesome. In many cases, the above methods are not suitable for treating many samples for many examination items. The method using a dry immunoassay element and a whole blood sample is not satisfied with accuracy. The methods using a biosensor or immunochromatography require particular equipment.
Accordingly, the object of the present invention is to provide an assaying means capable of rapidly measuring an analyte in a whole blood sample, using a whole blood as a sample without pretreatment of the blood. More particularly, the object is to provide an assaying means capable of obtaining a result for an analyte from a whole blood sample, identical to that from a conventional sample, such as serum or plasma, even when a problem associated with the whole blood sample, such as troublesome variation of existing ratio of insoluble components and soluble components, is encountered, to thereby enable a rapid examination without pretreatment of the collected whole blood sample, and extremely save labor in a general examination.